Abstract:Position 52 of iso-1-cytochrome c in the yeast Saccharomyces cerevisiae was systematically replaced with all possible amino acids to investigate the molecular basis of the "global suppressor" activity for the N52I mutation. Isogenic strains containing the variant proteins were made with a mixed oligonucleotide-directed mutagenesis technique in vivo. A relationship between thermodynamic protein stability and cellular protein levels was established by comparing direct spectroscopic measurements of cytochrome c l… Show more
“…In addition, the pathway in vivo might not be related to the pathways being examined in vitro, although these latter studies are essential for considering all viable options. Studies with CcP mutants placed back inside yeast cells and subsequent examination of the phenotypic consequences of these mutations in analogy to similar studies on cytochrome c , will eventually play a key role in understanding the reduction pathways in Nature.…”
Section: B Proposed Function Of the Tryptophan
Radical
In Electron Tr...mentioning
ContentsI. Introduction 705 II. General Principles 706 A. One Electron Oxidized Amino Acids Thus Far Identified in Proteins 706 B. Biosynthesis of (Modified) Amino Acid Radicals 706 C. Emerging General Chemical Properties of Enzymes Utilizing Protein Radicals for Catalysis 708 D. Methods To Examine Radical Dependent Reactions 710 III. Background on Ribonucleotide Reductases 710 IV. Class I Ribonucleotide Reductases 712 A. Formation of the Tyrosyl Radical 713 B. Function of the Tyrosyl Radical 714 C. Thiyl Radical Involvement in Catalysis 716 D. Evidence for Enzyme-Mediated Radical Chemistry Using Nucleotide Analogues 717 E. Structure 719 V. Class II Ribonucleotide Reductases 719 A. Exchange Reaction: Detection of the Elusive Thiyl Radical 719 B. Role of the Thiyl Radical in Catalysis 721 VI. Pyruvate Formate Lyase 722 A. Characterization of the Glycyl Radical 723 B. Formation of the Glycyl Radical 723 C. Catalytic Mechanism 724 D. Enzyme-Mediated Radical Chemistry with Pyruvate Analogues 727 VII. Anaerobic Ribonucleotide Reductase 728 A. Background 728 B. Requirement for a Glycyl Radical 728 C. Mechanism of Nucleotide Reduction 729 VIII. Cytochrome c Peroxidase 730 A. Formation of the Ferryl Heme/Tryptophan Cation Radical 730 B. Proposed Function of the Tryptophan Radical in Electron Transfer 731 IX. Prostaglandin H Synthase 733 A. Structure: A Tyrosine Residue Revealed 733 B. Proposed Role of the Tyrosyl Radical in Catalysis 734 X. Photosynthetic Oxygen Evolution 736 A. Background 736 B. Proposed Roles of Tyrosyl Radicals Y Z • and Y D • 737 C. Spectroscopic Studies on Y Z • and Y D • 738 D. Proposed Mechanisms for Water Oxidation and O 2 Evolution 739 XI. Galactose Oxidase 741 A. Different Redox States of Galactose Oxidase 741 B. Structure: A Modified Tyrosine Residue Revealed 742 C. Is the Oxidized Amino Acid Associated with Apo Oxidized GAO the Novel Ortho-Thiol-Substituted Tyrosine? 743 D. Catalytic Mechanism 743 XII. Quinoproteins 744 A. Copper-Dependent Amine Oxidases 744 B. Methylamine Dehydrogenase 749 XIII. Other Systems in Which Protein-Based Radicals Have Been Proposed or Detected 751 A. Bovine Liver Catalase 751 B. DNA Photolyase 751 C. Dopamine β Monooxygenase 752 XIV. Summary and Outlook 754 XV. Abbreviations 754 XVI. Acknowledgments 755 XVII. References 755 JoAnne Stubbe is the Novartis professor of Chemistry and Biology at the Massachusetts Institute of Technology. She received her undergraduate degree from the University of Pennsylvania working with Ed Thornton and did NSF-sponsored undergraduate research with Ed Trachtenberg at Clark University. These mentors played a strong role in her interest in physical organic chemistry. She received her Ph.D.
“…In addition, the pathway in vivo might not be related to the pathways being examined in vitro, although these latter studies are essential for considering all viable options. Studies with CcP mutants placed back inside yeast cells and subsequent examination of the phenotypic consequences of these mutations in analogy to similar studies on cytochrome c , will eventually play a key role in understanding the reduction pathways in Nature.…”
Section: B Proposed Function Of the Tryptophan
Radical
In Electron Tr...mentioning
ContentsI. Introduction 705 II. General Principles 706 A. One Electron Oxidized Amino Acids Thus Far Identified in Proteins 706 B. Biosynthesis of (Modified) Amino Acid Radicals 706 C. Emerging General Chemical Properties of Enzymes Utilizing Protein Radicals for Catalysis 708 D. Methods To Examine Radical Dependent Reactions 710 III. Background on Ribonucleotide Reductases 710 IV. Class I Ribonucleotide Reductases 712 A. Formation of the Tyrosyl Radical 713 B. Function of the Tyrosyl Radical 714 C. Thiyl Radical Involvement in Catalysis 716 D. Evidence for Enzyme-Mediated Radical Chemistry Using Nucleotide Analogues 717 E. Structure 719 V. Class II Ribonucleotide Reductases 719 A. Exchange Reaction: Detection of the Elusive Thiyl Radical 719 B. Role of the Thiyl Radical in Catalysis 721 VI. Pyruvate Formate Lyase 722 A. Characterization of the Glycyl Radical 723 B. Formation of the Glycyl Radical 723 C. Catalytic Mechanism 724 D. Enzyme-Mediated Radical Chemistry with Pyruvate Analogues 727 VII. Anaerobic Ribonucleotide Reductase 728 A. Background 728 B. Requirement for a Glycyl Radical 728 C. Mechanism of Nucleotide Reduction 729 VIII. Cytochrome c Peroxidase 730 A. Formation of the Ferryl Heme/Tryptophan Cation Radical 730 B. Proposed Function of the Tryptophan Radical in Electron Transfer 731 IX. Prostaglandin H Synthase 733 A. Structure: A Tyrosine Residue Revealed 733 B. Proposed Role of the Tyrosyl Radical in Catalysis 734 X. Photosynthetic Oxygen Evolution 736 A. Background 736 B. Proposed Roles of Tyrosyl Radicals Y Z • and Y D • 737 C. Spectroscopic Studies on Y Z • and Y D • 738 D. Proposed Mechanisms for Water Oxidation and O 2 Evolution 739 XI. Galactose Oxidase 741 A. Different Redox States of Galactose Oxidase 741 B. Structure: A Modified Tyrosine Residue Revealed 742 C. Is the Oxidized Amino Acid Associated with Apo Oxidized GAO the Novel Ortho-Thiol-Substituted Tyrosine? 743 D. Catalytic Mechanism 743 XII. Quinoproteins 744 A. Copper-Dependent Amine Oxidases 744 B. Methylamine Dehydrogenase 749 XIII. Other Systems in Which Protein-Based Radicals Have Been Proposed or Detected 751 A. Bovine Liver Catalase 751 B. DNA Photolyase 751 C. Dopamine β Monooxygenase 752 XIV. Summary and Outlook 754 XV. Abbreviations 754 XVI. Acknowledgments 755 XVII. References 755 JoAnne Stubbe is the Novartis professor of Chemistry and Biology at the Massachusetts Institute of Technology. She received her undergraduate degree from the University of Pennsylvania working with Ed Thornton and did NSF-sponsored undergraduate research with Ed Trachtenberg at Clark University. These mentors played a strong role in her interest in physical organic chemistry. She received her Ph.D.
“…Mutations at Asn 52 (N52) of iso-1-cytochrome c , located in the least stable N-yellow substructure of cytochrome c , have large effects on the global stability of iso-1-cytochrome c ( − ). The global stability can be decreased by up to 1.0 kcal/mol and increased by up to 5.0 kcal/mol relative to the wild-type (WT) protein depending on the amino acid substitution at position 52.…”
Thermodynamic communication between protein substructures has been investigated by determining the stabilizing effect of mutations at position 52 in the least stable, N-yellow, substructure of cytochrome c on the second least stable, Red, and most stable, Blue, substructures of the protein. A Lys 73 --> His (H73) variant of iso-1-cytochrome c, containing these mutations was used to measure the stability of the Red substructure of cytochrome c through the pH and guanidine hydrochloride (gdnHCl) dependence of the His 73-mediated alkaline conformational transition. The stability of the Blue substructure was measured by global unfolding with gdnHCl and increased by 1 to 3.5 kcal/mol versus the H73 variant. The data demonstrate that the increase in stability of the Red substructure is similar to the increase in global stability, consistent with upward propagation of stabilizing energy from less (N-yellow) to more stable (Red and Blue) protein substructures. The result also supports sequential rather than independent unfolding of the N-yellow and Red substructures of cytochrome c. The data indicate that a leucine at position 52 alters the nature of partial unfolding of the Red substructure, a surprising effect for a single-site mutation. For all variants, the thermodynamics of formation of the Lys 79 alkaline state, which does not unfold the entire Red substructure, shows less stabilization of the portion of the protein unfolded relative to the stabilization of the Blue substructure, indicating that propagation of energy between substructures is somewhat disrupted when unfolding does not correspond to a natural substructure.
“…Given the substructure bridging hydrogen bond of the His 26 side chain, these three mutation sites will also permit investigation of how changes in a buried hydrogen bond network impact a distant intersubstructural hydrogen bond (for distances between mutation sites, see Figure ). In addition, these mutations were chosen because all are known to significantly stabilize iso-1-cytochrome c ( − ), and structural data is available for iso-1-cytochrome c variants containing each or both of the N52I and Y67F mutations, as well as for the WT protein ( − ). 1 Yeast iso-1-cytochrome c in the oxidized state with the N52I and Y67F mutations, shown with the substructure classifications of horse cytochrome c according to refs 1 and 2.…”
mentioning
confidence: 99%
“…From thermodynamic studies, it is known that the N26H, N52I, and Y67F mutations all stabilize iso-1-cytochrome c ( − ) . In previous work, we also found a strong cooperative interaction between the N52I and N26H mutations ().…”
Cooperativity mediated through hydrogen bond networks in yeast iso-1-cytochrome c was studied using a thermodynamic triple mutant cycle. Three known stabilizing mutations, Asn 26 to His, Asn 52 to Ile, and Tyr 67 to Phe, were used to construct the triple mutant cycle. The side chain of His 26, a wild-type residue, forms two hydrogen bonds that bridge two substructures of the wild-type protein, and Tyr 67 and Asn 52 are part of an extensive buried hydrogen bond network. The stabilities of all variants in the triple mutant cycle were determined by guanidine hydrochloride denaturation methods and used to determine the pairwise, Delta(2)G(int), and triple interaction energies. His 26 and Ile 52 interact cooperatively (Delta(2)G(int) is 1-2 kcal/mol), whereas the two other pairs of mutations interact anticooperatively (Delta(2)G(int) is -0.5 to -1.5 kcal/mol). Previously reported structural data for iso-1-cytochrome c variants containing these mutations show that changes in the strength of the His 26 to Glu 44 hydrogen bond, apparently caused by changes in main chain dynamics, provide a mechanism for the long distance (His 26 to Phe 67 and His 26 to Ile 52) propagation of pairwise interaction energies. Opposing changes in the strength of the His 26 to Glu 44 hydrogen bond caused by the N52I and Y67F mutations generate a negative triple interaction energy (-0.9 +/-0.7 kcal/mol) that combined with cancellation of cooperative and anticooperative pairwise interactions produce apparent additivity for the stabilizing effects of the single mutations in the triple mutant variant.
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